Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

Abstract

Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

An Alternate Route for Adeno-associated Virus (AAV) Entry Independent of AAV Receptor

“…Although there is no observable role for AAVR in the cellular attachment of AAV4 and AAVrh32.33, we wanted to determine whether these vectors are able to bind to AAVR even if they are unable to functionally engage it for cellular entry. We used a previously reported virus overlay assay (34,46) to interrogate the binding of AAV4 to purified AAVR or AAVR expressed in cell lysates. The AAV4 virus overlay on purified AAVR domains illustrates that under the same conditions as those that demonstrated AAV2 binding to AAVR, no detectable binding of AAV4 to AAVR PKD1, PKD2, PKD3, or PKD1-5 is observed (Fig.…”

“…Recently a multiserotype AAV receptor (AAVR) was identified by using an unbiased genome-scale genetic screen (33). AAVR directly interacts with AAV particles via Ig-like polycystic kidney disease (PKD) domains present in the ectodomain of AAVR (33,34). It is required for the efficient transduction of multiple AAV serotypes (AAV1, -2, -3B, -5, -6, -8, and -9) in all cell lines that were tested.…”

Neuraminidase‐mediated desialylation augments AAV9‐mediated gene expression in skeletal muscle

“…The AAVR was shown to play an essential role in infection by multiple AAV serotypes (including AAV1, AAV2, AAV3b, AAV5, AAV6, AAV8 and AAV9) with respect to mediating rapid endocytosis from the plasma membrane and trafficking to the trans‐Golgi network. Subsequent work showed that AAVR is an N‐linked glycosylated membrane protein using the host cell endosomal pathway to cycle between the plasma membrane and the trans‐Golgi network . The finding of N‐linked glycosylation on the AAVR opens the possibility that it may function during both the initial attachment and the secondary internalization of AAV9 capsids.…”

“…Subsequent work showed that AAVR is an N-linked glycosylated membrane protein using the host cell endosomal pathway to cycle between the plasma membrane and the trans-Golgi network. 32 The finding of N-linked glycosylation on the AAVR opens the possibility that it may function during both the initial attachment and the secondary internalization of AAV9 capsids. This potential duality of function in the AAVR may help explain why ischemia and desialylation have such dramatic impacts on the efficiency of AAV9-mediated gene expression.…”

Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range

“…Other parvoviruses and adeno-associated viruses (AAV) use a variety of different receptors for binding and infection of cells. The AAVs use a protein receptor (the AAV receptor), which they bind through different domains, in addition to a variety of other glycoprotein or glycan receptors, including heparin sulfate proteoglycans and sialic acids (49)(50)(51). Those distinct interactions determine the tissue tropism of the AAVs and the gene delivery vectors derived from them (52).…”